Methylation regulation for FUNDC1 stability in childhood leukemia was up-regulated and facilitates metastasis and reduces ferroptosis of leukemia through mitochondrial damage by FBXL2

Abstract Leukemia, the most common malignant tumor in childhood, can be categorized into acute leukemia and chronic leukemia. However, the role of FUNDC1 in childhood leukemia (CL) remains unknown. This study aims to investigate the effects of FUNDC1 on patients with CL and its underlying mechanism both in vivo and in vitro. The mRNA expression levels of FUNDC1 were found to be up-regulated in serum samples from CL patients as well as in leukemia cell lines. Furthermore, it was observed that the mRNA expression of FUNDC1 was lower in stage I–II CL patients compared to stage III–IV patients. The up-regulation of FUNDC1 was found to promote leukemia metastasis. Additionally, it was discovered that FUNDC1 up-regulation reduces ferroptosis by inhibiting mitochondrial damage. In a leukemia model, FUNDC1 up-regulation induces the expression of FBXL2. Moreover, FUNDC1 up-regulation reduces FBXL2 ubiquitination, thus maintaining FBXL2 protein expression in leukemia. By inducing FBXL2, FUNDC1 reduces ferroptosis in leukemia through the inhibition of mitochondrial damage. The stability of FUNDC1 is controlled by METTL3 methylation. Overall, this study sheds light on the role of FUNDC1 in CL and provides insights into its underlying mechanisms.


Introduction
Leukemia, the most common malignant tumor in childhood, can be classified into acute leukemia (AL) and chronic leukemia [1].Among children, AL is more prevalent, with 80% of cases being acute lymphoblastic leukemia (ALL) [2].In China, the incidence of childhood leukemia (CL) is approximately 4/100,000, and it is increasing by 9,000 cases per year.Leukemia has a poor prognosis and is a leading cause of death in children [3].Therefore, understanding the epidemiological characteristics of CL is crucial for early diagnosis and treatment [3].
Leukemia is the most common malignant tumor disease in childhood.With the continuous progress of medical technology, the 5-year survival rate of leukemia patients has exceeded 80%.Leukemia develops into a chronic disease, and the number of cancer survivors increases accordingly [1].China usually defines cancer patients who have completed routine treatment and entered a stable followup period as cancer survivors [2].The main social need for cancer survivors in their school-age and adolescence is to return to school.However, these survivors may face the risk of delayed adverse reactions or health restrictions, which will affect the school adaptation of CL survivors [3].
Cell is a crucial process for maintaining normal growth, development, and internal environment stability in individuals.The classical modes of cell death include apoptosis, autophagy, and necrosis [4].Ferroptosis is a newly discovered form of cell death regulation characterized by cell volume shrinkage, increased mitochondrial membrane density, and reduced cristae [5].Unlike necrosis and apoptosis, ferroptosis is primarily characterized by the generation of reactive oxygen species (ROS), lipid peroxidation, and iron accumulation [6].Recent research on the mechanism of ferroptosis has shown its close association with the occurrence and progression of tumors [6,7].Leukemia and lymphoma, as malignant tumors of the hematopoietic system, have low efficacy with existing treatment plans, necessitating exploration of new treatment models [8].The advancements in ferroptosis research have provided novel insights for the treatment of hematopoietic system tumors [9].
As one of the core regulatory receptors for damaged mitochondrial clearance, FUN14 domain containing (1) (FUNDC1) plays an important protective role in myocardial injury [10].FUNDC1 is a protein located in the outer membrane of mitochondria, which participates in mitochondrial fusion and division to maintain heart function.The absence of FUNDC1 can lead to heart failure.Under normal circumstances, FUNDC1 interacts with the mitochondrial fusion protein optic atrophy protein 1 located on the inner membrane of mitochondria to provide energy for myocardial cells [11,12].Under stress conditions, FUNDC1 can promote the translocation of mitochondrial related protein 1 to the mitochondrial outer membrane, thereby inducing mitochondrial division.FUNDC1 is expressed in various cells, tissues, or organs and can recruit LC3 to regulate cell autophagy through its unique Leucine-rich repeat conserved domain.There is evidence that the abnormal expression of FUNDC1 is involved in the pathological progression of various cancer diseases and can serve as a potential target [11,12].However, the role of FUNDC1 in CL is unknown.This study investigated the effects of FUNDC1 on patients with CL and its underlying mechanism in vivo and in vitro.

Patients with CL
This study was approved by the Ethics Committee of our hospital.All the serum samples were immediately snap frozen in liquid nitrogen and stored at −80°C for further using.Pathological evaluation was performed according to the WHO classification by two experienced clinical pathologists.
Inclusion criteria were (1) meets the diagnostic criteria related to childhood ALL, (2) age <12 years old, (3) family members have informed consent to the treatment method and related precautions, and have good compliance, (4) complete clinical data, and (5) the resident population of this region.
Exclusion criteria included: (1) intolerance to the medication used in this study, (2) after diagnosis, without treatment or voluntarily giving up treatment or transferring to another hospital during the treatment period, (3) received corresponding treatment before enrollment, (4) concomitant systemic acute and chronic infections, and (5) severe liver and kidney dysfunction.
Ethical approval: The current study was approved by the Ethics Committee of Hubei Enshi Tujia and Miao Autonomous Prefecture Central Hospital and complied with the guidelines outlined in the declaration of Helsinki were followed.
Informed consent: Informed consent was obtained from all participants.

Quantitative polymerase chain reaction (qPCR)
Total RNAs were isolated with RNA isolator total RNA extraction reagent (Takara) and cDNA was synthesized using PrimeScipt RT Master Mix (Takara).qPCR were performed with the ABI Prism 7500 sequence detection system according to the Prime-ScriptTM RT detection kit.Relative levels of the sample mRNA expression were calculated and expressed as 2 −△△Ct .

Proliferation assay and EDU staining
For cell counting kit-8, after 48 h of transfection, a total of approximately 2 × 10 3 cells/well was seeded into 96-well plate.After culturing at indicated time, the cellular proliferation was detected using CellTiter-GloR Luminescent Cell Viability Assay (Beyotime) according to manufacturer's instructions.
For ethynyl deoxyuridine (EdU) incorporation assay, EdU (10 mM) was added to each well and cells were fixed with 4% formaldehyde for 30 min.After washing, EdU was detected with Click-iTR EdU Kit and images were visualized using fluorescent microscope (Olympus).

Statistical analyses
Graphad Prism 6 was used for the statistical analysis.P < 0.05 was considered statistically significant.Comparisons of data between groups were followed using Student's t-test or one-way analysis of variance, followed by Tukey's post hoc test.

Expression levels of FUNDC1 in CL
To investigate the potential role of FUNDC1 in CL patients, we examined the expression levels of FUNDC1 using PCR.Our data revealed that the mRNA expression levels of FUNDC1 were up-regulated in serum samples from CL patients, compared to normal group (Figure 1a).Furthermore, the mRNA expression of FUNDC1 was lower in I-II stage CL patients compared to III-IV stage patients, with an receiver operating characteristic (ROC) value of 0.8536 (Figure 1b and c).Meanwhile, FUNDC1 mRNA and protein expression levels were also up-regulated in leukemia cell lines, compared with HS-5 cell (Figure 1d and e).We also observed an increase in FUNDC1 protein expression in 6T-CEM cells (Figure 1e).These findings suggest that FUNDC1 expression is increased in leukemia and may play a role in the progression of the disease.

FUNDC1 up-regulation promotes leukemia metastasis in in vitro model of leukemia
To investigate the role of FUNDC1 in leukemia cell metastasis, we conducted an experiment where we overexpressed FUNDC1 in leukemia cells using a FUNDC1 plasmid (Figure 2a).Our results demonstrated that up-regulation of FUNDC1 significantly enhanced cell proliferation, as indicated by an increase in EDU-positive cells, and increased the migration rate of leukemia cells (Figure 2b-d).On the other hand, when we knocked down FUNDC1 using siRNA, we observed a decrease in FUNDC1 mRNA expression in leukemia cells (Figure 2e), which subsequently inhibited cell proliferation, as indicated by a decrease in EDU-positive cells, and reduced the migration rate (Figure 2f-h).These findings suggest that up-regulation of FUNDC1 promotes cell proliferation and metastasis in leukemia.Further validation using fluorescence microscopy confirmed the reduction in cell proliferation under FUNDC1 knockdown conditions, as evidenced by fewer EDU-positive cells (Figure 2i).Additionally, microscopy images depicted reduced cellular migration, supporting the quantitative data (Figure 2j).These findings collectively suggest that the up-regulation of FUNDC1 promotes cell proliferation and metastasis in leukemia.

FUNDC1 up-regulation reduces ferroptosis by the inhibition of mitochondrial damage in in vitro model of leukemia
In addition, we delved into the role of FUNDC1 in ferroptosis using an in vitro model of leukemia.Our findings revealed that the up-regulation of FUNDC1 led to an increase in JC-1 levels and mitochondrial permeability transition (MPT) as determined by the calcein AM/CoCl2 assay.It also resulted in a decrease in PI-positive cells and the activity levels of caspase-3/9.Moreover, FUNDC1 up-regulation inhibited the activity levels of lactate dehydrogenase (LDH) and the iron content in leukemia cells.It expanded the levels of glutathione (GSH) and increased the protein expression of GPX4 (Figure 3a-i).Conversely, knockdown of FUNDC1 reduced JC-1 levels and MPT, increased PI-positive cells and caspase-3/9 activity levels, promoted LDH activity levels and iron content, suppressed GSH levels, and decreased GPX4 protein expression in leukemia cells (Figure 3j-q).These data suggest that the up-regulation of FUNDC1 mitigates ferroptosis by inhibiting mitochondrial damage in leukemia cells.

FUNDC1 up-regulation induces FBXL2 expression in in vitro model of leukemia
The study further investigated the role of FBXL2 in mediating the effects of FUNDC1 on ferroptosis in leukemia through mitochondrial damage.We observed that the upregulation of FUNDC1 resulted in an increase in FBXL2 mRNA expression, while si-FUNDC1 suppressed FBXL2 mRNA expression in leukemia cells (Figure 4a).Furthermore, FBXL2 up-regulation suppressed the protein expression of both FBXL2 and FUNDC1 in leukemia cells (Figure 4b), whereas FBXL2 down-regulation led to a decrease in the protein expression of FBXL2 and FUNDC1 (Figure 4c).
Confocal microscopy analysis revealed that FUNDC1 upregulation increased the expression of both FUNDC1 and FBXL2 in leukemia cells (Figure 4d).Additionally, immunoprecipitation experiments demonstrated that FBXL2 protein interacted with FUNDC1 protein (Figure 4e).Moreover, we found that the up-regulation of FUNDC1 reduced FBXL2 ubiquitination, thereby maintaining FBXL2 protein expression in leukemia cells (Figure 4f).Collectively, these findings suggest that FUNDC1 forms a complex with FBXL2 to attenuate FBXL2 ubiquitination in the leukemia model.

Mitochondrial damage regulates the effects of FUNDC1 up-regulation on ferroptosis and metastasis in in vitro model of leukemia
We investigated the functional involvement of mitochondrial damage in the effects of FUNDC1 on ferroptosis in leukemia.To induce mitochondrial damage, we treated leukemia cells with a mitochondrial damage activator, BMS-191095, at a concentration of 20 μmol/L.As a result, we observed a reduction in JC-1 level and MPT, an increase in PI positive cells and LDH activity level, and a decrease in GSH level and GPX4 protein expression in leukemia cells with FUNDC1 up-regulation (Figure 5a-h).Conversely, when we used a mitochondrial damage inhibitor, olesoxime, at a concentration of 10 µM, we observed an increase in JC-1 level and MPT, a decrease in PI positive cells and caspase-3/9 activity levels, a decrease in LDH activity level, and an increase in GSH level and GPX4 protein expression in leukemia cells with FUNDC1 down-regulation (Figure 5i-p).These findings suggest that mitochondrial damage is functionally involved in the effects of FUNDC1 on ferroptosis in leukemia.Next, we further investigated the impact of mitochondrial damage on leukemia cells with FUNDC1 upregulation or down-regulation.Treatment with the mitochondrial damage activator led to a significant decrease in cell proliferation, as demonstrated by reduced EDU positive cells, and a decreased migration rate in leukemia cells with FUNDC1 up-regulation (Figure 6a-c).Conversely, treatment with the mitochondrial damage inhibitor resulted in enhanced cell proliferation, as indicated by increased EDU positive cells, and an increased migration rate in leukemia cells with FUNDC1 down-regulation (Figure 6d-f).These findings strongly suggest that FUNDC1 exerts its protective effect against ferroptosis in leukemia by inhibiting mitochondrial damage through the induction of FBXL2.

Methylation controls FUNDC1 stability in in vitro model of leukemia
The FUNDC1 gene contains several potential methylation modification sites near its stop codon, as shown in Figure 7a.In vitro models of leukemia treated with si-METTL3, an m6A antibody, resulted in a decrease in FUNDC1 mRNA enrichment levels (Figure 7b).Si-METTL3 also decreased the stability of FUNDC1 mRNA in these in vitro leukemia models (Figure 7c).Four m6A sites were identified in the 3ʹ-untranslated region of FUNDC1, with significant enrichment observed at sites 1, 2, 3, and 4 (Figure 7d).Si-METTL3 reduced the luciferase activity levels of wild-type FUNDC1 and two of its sites (Figure 7e).Furthermore, m6A enrichment increased the levels of FUNDC1 at sites 1, 2, 3, and 4 (Figure 7f), while si-METTL3 reduced the m6A levels of FUNDC1 at these sites (Figure 7g).These findings indicate that METTL3 methylation plays a role in controlling the stability of FUNDC1.

Discussion
Leukemia is the most prevalent cancer in children, accounting for approximately 35% of all childhood malignancies.It has become a chronic, non-communicable disease that poses a serious threat to children's health [13,14].CL differs significantly from adult leukemia in terms of disease spectrum composition, morphology, immunology, cytogenetics, molecular biology, and clinical prognosis.This indicates that CL has specific characteristics in terms of its pathogenesis, epidemiology, and other aspects [15].In this study, the mRNA expression levels of FUNDC1 were up-regulated in serum samples from CL patients and leukemia cell lines.Hou et al. suggested that FUNDC1 might be a prognostic biomarker in patients with cervical cancer [16].So, these results showed that FUNDC1 participated in disease progression of CL.In this study, we only collected 40 patients, which is insufficient, and is also the weakness of this study.We hope to collect more patients in the next experiment.ALL is the most prevalent malignant hematological disorder among children in China, making up around 65-70% of all cases of childhood AL [17].Childhood ALL is currently one of the malignancies with the most successful treatment outcomes and highest rates of cure, with a 5-year event-free survival rate exceeding 80% [18].We found that FUNDC1 up-regulation promotes leukemia metastasis.Hui et al. showed that FUNDC1 regulated laryngeal cancer cells survival [19].Meanwhile, this study only used cell lines, which is insufficient, we will use more models in further experiment.
Iron is a crucial metal ion involved in various metabolic processes in the human body and plays a significant role in promoting ferroptosis [20,21].Exosomes released by cells treated with GPX4 inhibitors contain high levels of ferritin [20].During ferroptosis, the level of prominin 2 is inversely correlated with the amount of free iron in the cell, suggesting that exosomes can protect cells from ferroptosis by eliminating iron from the cell [6].Ferroptosis is accompanied by the accumulation of ROS and an increase in lipid peroxidation levels.GPX4 mitigates the susceptibility of cells to ferroptosis by reducing ROS accumulation through its antioxidant activity; consequently, the level and activity of GPX4 can influence the extent of ferroptosis [7].Moreover, system X-plays a critical role in ferroptosis by regulating the ROS balance through its impact on GSH metabolism [22].These data of this study showed that FUNDC1 reduces ferroptosis in leukemia through the inhibition of mitochondrial damage by the induction of FBXL2.Bi et al. favored that FUNDC1 interacts with GPX4 to trigger ferroptosis in model of liver fibrosis through mitochondrial translocation [23].
In conclusion, this study demonstrates that FUNDC1 promotes metastasis and reduces ferroptosis in a leukemia model through FBXL2 activation.These findings provide a new mechanism for understanding the inhibition of FUNDC1 in leukemia and suggest a novel target for cancer treatment.Therefore, FUNDC1 is a potential target for the treatment of various types of cancer in further clinical applications.

Figure 1 :
Figure 1: Expression levels of FUNDC1 in CL.FUNDC1 mRNA expression (a) and (b), ROC (c) in patients with CL; FUNDC1 mRNA and protein expression (d) and (e) in leukemia cells.Normal, normal serum sample; Cancer, patients with CL; **p < 0.01 compared with normal serum sample or HS-5 cells.

Figure 2 :
Figure 2: FUNDC1 up-regulation promotes leukemia metastasis.FUNDC1 mRNA expression (a), cell growth (b), metastasis (c), and EDU assay (d) in vitro model of leukemia by FUNDC1 up-regulation; FUNDC1 mRNA expression (e), cell growth (f), metastasis (g), and EDU assay (h) in vitro model of leukemia by FUNDC1 down-regulation.(i).Fluorescence images showing reduced proliferation in leukemia cells with FUNDC1 knockdown, as evidenced by fewer EDU-positive cells.(j).Micrographs illustrating decreased migration of leukemia cells after FUNDC1 knockdown.Vector, negative control group; FUNDC1, over-expression of FUNDC1 group; Si-nc, si-negative control group; Si-FUNDC1, down-regulation of FUNDC1 group; **p < 0.01 compared with negative control group or si-negative control group.

Figure 6 :
Figure 6: Mitochondrial damage regulates the effects of FUNDC1 up-regulation on metastasis in leukemia.Cell growth (a), metastasis (b), and EDU assay (c) in vitro model of leukemia by FUNDC1 up-regulation; cell growth (d), metastasis (e), and EDU assay (f) in vitro model of leukemia by FUNDC1 down-regulation.Vector, negative control group; FUNDC1, over-expression of FUNDC1 group; Si-nc, si-negative control group; Si-FUNDC1, downregulation of FUNDC1 group; **p < 0.01 compared with negative control group or si-negative control group.